Dimeric reverse transcriptase

ABSTRACT

Covalently-linked DNA polymerases are provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present patent application is a divisional of U.S. Ser. No.15/370,646, filed Dec. 6, 2016, now U.S. Pat. No. 10,364,420, whichclaims benefit of priority to U.S. Provisional Patent Application No.62/264,223, filed on Dec. 7, 2015, which is incorporated by referencefor all purposes.

REFERENCE TO A “SEQUENCE LISTING” SUBMITTED AS ASCII TEXT FILES VIAEFS-WEB

The Sequence Listing written in file Seq-List.txt created on Jul. 29,2019, 196 KB, machine format IBM-PC, MS-Windows operating system, inaccordance with 37 C.F.R. §§ 1.821- to 1.825, is hereby incorporated byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

The detection, analysis, transcription, and amplification of nucleicacids are frequently-used procedures in modern molecular biology. DNApolymerases are useful for detection and amplification of DNA or RNA.The application of such procedures for RNA analysis can involve theinvestigation of gene expression, diagnosis of infectious agents orgenetic diseases, and the generation of cDNA, to name but a fewapplications. The reverse transcription (“RT”) of RNA thus has manyuses. In some instances, the RT is followed by polymerase chain reactionamplification which can be used for rapid detection and quantificationof RNA. This procedure is often referred to as “RT-PCR”.

BRIEF SUMMARY OF THE INVENTION

Polypeptides comprising at least two DNA polymerases covalently linkedtogether are provided. Such polypeptides have increased stability andcan have reduced reaction times compared to monomer DNA polymerases. Insome embodiments, a polypeptide comprising a first DNA polymerase and asecond DNA polymerase covalently linked by a heterologous linker isprovided. In some embodiments, the first DNA polymerase is a firstreverse transcriptase and the second DNA polymerase is a second reversetranscriptase.

In some embodiments, the polypeptide is a fusion protein and theheterologous linker is an amino acid linker that links the carboxylterminus of the first reverse transcriptase to the amino terminus of thesecond reverse transcriptase. In some embodiments, the polypeptidefurther comprises a third reverse transcriptase covalently linked by asecond linker to the first or second reverse transcriptase.

In some embodiments, the polypeptide is no more than 1400, 1500, 1600,1700, 1800, 1900 or 2000 amino acids in length.

In some embodiments, the first reverse transcriptase and the secondreverse transcriptase are identical. In some embodiments, the firstreverse transcriptase and the second reverse transcriptase are at least80%, 85%, 90%, or 95% identical. In some embodiments, the first andsecond reverse transcriptases are a murine leukemia virus (MLV) reversetranscriptase. In some embodiments, the first and second reversetranscriptases are a Feline leukemia virus (FLV) reverse transcriptase.

In some embodiments, the first reverse transcriptase and the secondreverse transcriptase are less than 70%, 75%, 80%, 95% or 90% identical.

In some embodiments, the amino acid linker is between 1-30 or 1-50(e.g., 1-15, 3-25) amino acids long.

In some embodiments, the first or second reverse transcriptase isselected from the group consisting of murine leukemia virus (MLV)reverse transcriptase, Feline leukemia virus (FLV) reversetranscriptase, bovine leukemia virus (BLV), Avian Myeloblastosis Virus(AMV) reverse transcriptase, Respiratory Syncytial Virus (RSV) reversetranscriptase, Equine Infectious Anemia Virus (EIAV) reversetranscriptase, Rous-associated Virus-2 (RAV2) reverse transcriptase,SUPERSCRIPT II reverse transcriptase, SUPERSCRIPT I reversetranscriptase, THERMOSCRIPT reverse transcriptase and MMLV RNase H—reverse transcriptase.

In some embodiments, the first reverse transcriptase is a murineleukemia virus (MLV) reverse transcriptase and the second reversetranscriptase is a Feline leukemia virus (FLV) reverse transcriptase. Insome embodiments, the first reverse transcriptase is a Feline leukemiavirus (FLV) reverse transcriptase and the second reverse transcriptaseis a murine leukemia virus (MLV) reverse transcriptase.

In some embodiments, at least the first or the second reversetranscriptase have at least one mutation compared to anaturally-occurring reverse transcriptase. In some embodiments, thefirst or the second reverse transcriptase is an RNase H— reversetranscriptase.

In some embodiments, the first DNA polymerase is a first DNA-dependentpolymerase and/or the second DNA polymerase is a second DNA-dependentpolymerase. In some embodiments, the first DNA polymerase is aDNA-dependent polymerase and the second DNA polymerase is a reversetranscriptase. In some embodiments, the first DNA polymerase is areverse transcriptase and the second DNA polymerase is a DNA-dependentpolymerase.

Also provided is nucleic acid (optionally isolated or purified) encodinga polypeptide as described above or elsewhere herein. Also provided is apolynucleotide vector comprising the nucleic acid as described herein.Also provided is a host cell (e.g., a prokaryotic, fungal, yeast, oreukaryotic cell) comprising the vector.

Also provided is a reaction mixture comprising: purified mRNA; and thepolypeptide as described above or elsewhere herein. In some embodiments,the reaction mixture further comprises a buffer selected from the groupconsisting of Tris, HEPES, ACES, PIPES, MOPSO, BES, MOPS, TES, TAPSO,POPSO, BICINE, TAPS, and AMPSO. In some embodiments, the reactionmixture further comprises at least one oligonucleotide primer and/ordeoxynucleotides.

Also provided is a kit comprising the polypeptide as described above orelsewhere herein. In some embodiments, the polypeptide comprises one ortwo reverse transcriptases and the kit further comprises a DNA-dependentDNA polymerase.

Also provided is a method of performing a polymerase reaction. In someembodiments, the method comprises, contacting a target nucleic acid tothe polypeptide as described above of elsewhere herein with anoligonucleotide primer that hybridizes to the target nucleic acid underconditions to allow the polypeptide to extend the oligonucleotide primerin a template-dependent manner, thereby performing a polymerasereaction. In some embodiments, the target nucleic acid is a RNA and thepolymerase comprises one (or two) reverse transcriptases. In someembodiments, the target nucleic acid is a DNA and the polymerasecomprises one (or two) DNA-dependent DNA polymerases.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization described below are those well known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. The techniques and procedures are generallyperformed according to conventional methods in the art and variousgeneral references (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, andorganic synthetic described below are those well known and commonlyemployed in the art. Standard techniques, or modifications thereof, areused for chemical syntheses and chemical analyses.

“Heterologous”, when used with reference to portions of a protein,indicates that the protein comprises two or more domains that are notfound in the same relationship to each other in nature. Such a protein,e.g., a fusion protein, contains two or more sequences covalently linkedvia a peptide bond or peptide linker sequence arranged to make a newfunctional protein.

A “primer” refers to a polynucleotide sequence that hybridizes to asequence on a target nucleic acid and serves as a point of initiation ofnucleic acid synthesis. Primers can be of a variety of lengths and areoften less than 50 nucleotides in length, for example 12-30 nucleotides,in length. The length and sequences of primers for use in PCR can bedesigned based on principles known to those of skill in the art, see,e.g., Innis et al., supra.

“Polymerase” refers to an enzyme that performs template-directedsynthesis of polynucleotides. The term encompasses both the full lengthpolypeptide and a domain that has polymerase activity.

A “template” refers to a polynucleotide sequence that comprises thepolynucleotide to be amplified, optionally flanked by one or two primerhybridization sites.

As used herein, “nucleic acid” means DNA, RNA, single-stranded,double-stranded, or more highly aggregated hybridization motifs, and anychemical modifications thereof. Modifications include, but are notlimited to, those providing chemical groups that incorporate additionalcharge, polarizability, hydrogen bonding, electrostatic interaction,points of attachment and functionality to the nucleic acid ligand basesor to the nucleic acid ligand as a whole. Such modifications include,but are not limited to, peptide nucleic acids (PNAs), phosphodiestergroup modifications (e.g., phosphorothioates, methylphosphonates),2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, methylations, unusual base-pairing combinationssuch as the isobases, isocytidine and isoguanidine and the like. Nucleicacids can also include non-natural bases, such as, for example,nitroindole. Modifications can also include 3′ and 5′ modifications suchas capping with a fluorophore (e.g., quantum dot) or another moiety.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., a carbon atom thatis bound to a hydrogen atom, a carboxyl group, an amino group, and an Rgroup, e.g., homoserine, norleucine, methionine sulfoxide, methioninemethyl sulfonium. Such analogs have modified R groups (e.g., norleucine)or modified peptide backbones, but retain the same basic chemicalstructure as a naturally occurring amino acid. Amino acid mimeticsrefers to chemical compounds that have a structure that is differentfrom the general chemical structure of an amino acid, but that functionsin a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The term “promoter” refers to regions or sequence located upstreamand/or downstream from the start of transcription and which are involvedin recognition and binding of RNA polymerase and other proteins toinitiate transcription.

A “vector” refers to a polynucleotide, which when independent of thehost chromosome, is capable replication in a host organism. Preferredvectors include plasmids and typically have an origin of replication.Vectors can comprise, e.g., transcription and translation terminators,transcription and translation initiation sequences, and promoters usefulfor regulation of the expression of the particular nucleic acid.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Accelrys), or by manual alignment and visualinspection.

Percent sequence identity and sequence similarity is determined usingthe BLAST algorithm, which is described in Altschul et al., J. Mol.Biol. 215:403-410 (1990). Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (see Worldwide Website: ncbi.nlm.nih.go-v/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al, supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl.Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

It has been surprisingly discovered that covalent linkage of two DNApolymerases results in a polypeptide that is more thermostable and insome cases has an improved (shorter) reaction time compared to monomerpolymerases. For instance, as shown in the Examples, protein fusions ofthe same or different reverse transcriptases result in polypeptides withmore heat stability and in some cases lower reaction times than acontrol monomeric reverse transcriptase.

II. Dimeric Proteins

Dimeric (and trimeric) DNA polymerases are provided herein. Suchpolypeptide can be isolated or purified or can be in complex mixturesor, for example, in cells. The polypeptides as described herein cancomprise, for example, at least two and in some embodiments, threedifferent DNA polymerases. The different DNA polymerases can beidentical or they can have different sequences.

For convenience, in a dimeric polypeptide, the DNA polymerase nearest atthe amino terminus of the polypeptide is referred to as the “first” DNApolymerase and the DNA polymerase nearest the carboxyl terminus isreferred to as the “second” DNA polymerase. In cases where thepolypeptide is a trimer, the middle DNA polymerase is the “second” andthe DNA polymerase nearest the carboxyl terminus of the polypeptide isthe “third” DNA polymerase. “DNA polymerase” refers to any protein(full-length as occurs in nature or a fragment or variant thereof)having DNA polymerase activity. Similarly, a “reverse transcriptase”refers to any protein (full-length as occurs in nature or a fragment orvariant thereof) having reverse transcriptase activity

In some embodiments one or both of the DNA polymerases in thepolypeptide (or in the case of trimers, one, both, or all three) can bereverse transcriptases, i.e., an RNA-dependent DNA polymerase. In someembodiments, the polypeptides described herein comprise two reversetranscriptases. In embodiments in which the polypeptide comprises tworeverse transcriptases, the reverse transcriptases can be identical insequence or the reverse transcriptases can have different sequences. Insome embodiments, the two reverse transcriptases are at least 80%, 85%,90%, 95%, or 99% identical to each other. In other embodiments, the tworeverse transcriptases are less than 70%, 80%, or 90% (e.g., 40-70,40-80, 40-90%) identical to each other. For example, two differentreverse transcriptases (or one reverse transcriptase and oneDNA-dependent DNA polymerase) can be selected with complementary butdifferent activities such that the polypeptide comprising the tworeverse transcriptases (or one RT and one DNA-dependent DNA polymerase)has superior activity compared to either of the individual monomericenzymes.

Exemplary reverse transcriptases that can be the first or second (orthird) reverse transcriptase include, but are not limited to, murineleukemia virus (MLV) reverse transcriptase (Gerard and Grandgenett,Journal of Virology 15:785-797, 1975; Verma, Journal of Virology15:843-854, 1975) or SEQ ID NO:1, feline leukemia virus (FLV) reversetranscriptase (Rho and Gallo, Cancer Lett., 10:207-221, 1980 or SEQ IDNO:1, bovine leukemia virus (BLV) (Demirhan et al., Anticancer Res.,16:2501-5, 1996; Drescher et al., Arch Geschwulstforsch., 49:569-79,1979), Avian Myeloblastosis Virus (AMV) reverse transcriptase,Respiratory Syncytial Virus (RSV) reverse transcriptase, EquineInfectious Anemia Virus (EIAV) reverse transcriptase, Rous-associatedVirus-2 (RAV2) reverse transcriptase, SUPERSCRIPT II reversetranscriptase, SUPERSCRIPT III reverse transcriptase (U.S. Pat. Nos.8,541,219, 7,056,716, 7,078,208), THERMOSCRIPT reverse transcriptase andMMLV RNase H— reverse transcriptase.

In some one or both of the DNA polymerases (for example the reversetranscriptases) is a native polymerase. In other embodiments, one orboth of the DNA polymerases (for example the reverse transcriptases)comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-20, 1-10,1-5, 1-2) mutation compared to the native DNA polymerase (including butnot limited to SEQ ID NO:1 or SEQ ID NO:2). Exemplary mutations include,but are not limited to, mutations that reduce (e.g., reduce by at least50, 70, or 95%) or eliminate RNase H activity (referred to as “RNase H⁻mutations). Mutations that increase thermostability in RT can also beintroduced. Exemplary mutations of this type are described in, forexample, Arezi et al., Nucleic Acids Res. 2009 February; 37(2): 473-481.

In some embodiments, the polymerase comprises at least two (e.g., 2 or3) DNA-dependent DNA polymerases. Exemplary DNA-dependent DNA polymeraseinclude, but are not limited to, any of the polymerases of the fivefamilies of DNA-dependent DNA polymerases, although most will fall intofamilies A, B and C. There is little or no structural or sequencesimilarity among the various families. Most family A polymerases aresingle chain proteins that can contain multiple enzymatic functionsincluding polymerase, 3′ to 5′ exonuclease activity and 5′ to 3′exonuclease activity. Family B polymerases typically have a singlecatalytic domain with polymerase and 3′ to 5′ exonuclease activity, aswell as accessory factors. Family C polymerases are typicallymulti-subunit proteins with polymerizing and 3′ to 5′ exonucleaseactivity. In E. coli, three types of DNA polymerases have been found,DNA polymerases I (family A), II (family B), and III (family C). Ineukaryotic cells, three different family B polymerases, DNA polymerasesa, 6, and c are implicated in nuclear replication, and a family Apolymerase, polymerase γ, is used for mitochondrial DNA replication.Exemplary DNA-dependent polymerases can include, but are not limited to,Taq polymerase or iProof DNA polymerases. See, e.g., U.S. Pat. No.8,916,352.

Optionally, the DNA polymerase(s) can be linked to a non-specific DNAbinding domain. Examples of such proteins include, but are not limitedto, the Archaeal small basic DNA binding proteins Sso7d and Sso7d-likeproteins (see, e.g., Choli et al., Biochimica et Biophysica Acta950:193-203, 1988; Baumann et al., Structural Biol. 1:808-819, 1994; andGao et al, Nature Struc. Biol. 5:782-786, 1998), Archaeal HMf-likeproteins (see, e.g., Stanch et al., J. Molec. Biol. 255:187-203, 1996;Sandman et al., Gene 150:207-208, 1994), and PCNA homologs (see, e.g.,Cann et al., J. Bacteriology 181:6591-6599, 1999; Shamoo and Steitz,Cell: 99, 155-166, 1999; De Felice et al., J. Molec. Biol. 291, 47-57,1999; and Zhang et al., Biochemistry 34:10703-10712, 1995).

Sso7d and Sso7d-like proteins, Sac7d and Sac7d-like proteins, e.g.,Sac7a, Sac7b, Sac7d, and Sac7e are small (about 7,000 kd MW), basicchromosomal proteins from the hyperthermophilic archaebacteriaSulfolobus solfataricus and S. acidocaldarius, respectively. Theseproteins are lysine-rich and have high thermal, acid and chemicalstability. They bind DNA in a sequence-independent manner and whenbound, increase the T_(m) of DNA by up to 40° C. under some conditions(McAfee, Biochemistry 34:10063-10077, 1995; Gao et al., Nat. Struct.Biol. 5(9):782-786, 1998). These proteins and their homologs aretypically believed to be involved in stabilizing genomic DNA at elevatedtemperatures. Suitable Sso7d-like DNA binding domains for use in theinvention can be modified based on their sequence homology to Sso7d.Typically, DNA binding domains that are identical to or substantiallyidentical to a known DNA binding protein over a comparison window ofabout 25 amino acids, optionally about 50-100 amino acids, or the lengthof the entire protein, can be used in the invention. The sequence can becompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the describedcomparison algorithms or by manual alignment and visual inspection. Avariety of mutations in the Sso7 binding domain have been described in,e.g., US Patent Application Nos. 2005/0048530 and 2007/0141591.

The polypeptides can include additional amino acid sequences besidesthat of the two (or three) DNA polymerases and the linker(s). Forexample, in some embodiments, the polypeptide comprises additionalsequence at the amino or carboxyl terminus of the polypeptide. Examplesof such sequences include, but are not limited to, sequences foraffinity purification of the polypeptide, or fluorescent proteins. Inaddition, in some embodiments, the polypeptide can include one or morefluorescent label or a fluorescent label/quencher pair.

III. Linkers

Two DNA polymerases as described can be joined via a linker by methodswell known to those of skill in the art. These methods can includeeither chemical and recombinant means.

Chemical linking can be performed, for example, as described inBioconjugate Techniques, Hermanson, Ed., Academic Press (1996). Joiningcan include, for example, derivitization for the purpose of linking thetwo proteins to each other, either directly or through a linkingcompound, by methods that are well known in the art of proteinchemistry. For example, in one chemical conjugation embodiment, themeans of linking the catalytic domain and the nucleic acid bindingdomain comprises a heterobifunctional-coupling reagent which ultimatelycontributes to formation of an intermolecular disulfide bond between thetwo moieties. Other types of coupling reagents that are useful in thiscapacity for the present invention are described, for example, in U.S.Pat. No. 4,545,985. Alternatively, an intermolecular disulfide mayconveniently be formed between cysteines in each moiety, which occurnaturally or are inserted by genetic engineering. The means of linkingmoieties may also use thioether linkages between heterobifunctionalcrosslinking reagents or specific low pH cleavable crosslinkers orspecific protease cleavable linkers or other cleavable or noncleavablechemical linkages. Other chemical linkers include carbohydrate linkers,lipid linkers, fatty acid linkers, polyether linkers, e.g., PEG, etc.For example, poly(ethylene glycol) linkers are available from ShearwaterPolymers, Inc. Huntsville, Ala. These linkers optionally have amidelinkages, sulfhydryl linkages, or heterobifunctional linkages.

Linking two DNA polymerases may also comprise a peptidyl bond formedbetween moieties that are separately synthesized by standard peptidesynthesis chemistry or recombinant means. The conjugate protein itselfcan also be produced using chemical methods to synthesize an amino acidsequence in whole or in part. For example, peptides can be synthesizedby solid phase techniques, such as, e.g., the Merrifield solid phasesynthesis method, in which amino acids are sequentially added to agrowing chain of amino acids (see, Merrifield (1963) J. Am. Chem. Soc.,85:2149-2146). Equipment for automated synthesis of polypeptides iscommercially available from suppliers such as PE Corp. (Foster City,Calif.), and may generally be operated according to the manufacturer'sinstructions. The synthesized peptides can then be cleaved from theresin, and purified, e.g., by preparative high performance liquidchromatography (see Creighton, Proteins Structures and MolecularPrinciples, 50-60 (1983)). The composition of the synthetic polypeptidesor of subfragments of the polypeptide, may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure; seeCreighton, Proteins, Structures and Molecular Principles, pp. 34-49(1983)).

In addition, nonclassical amino acids or chemical amino acid analogs canbe introduced as a substitution or addition into the sequence.Non-classical amino acids include, but are not limited to, the D-isomersof the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxy-proline, sarcosine, citrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Cα-methyl amino acids, N-methyl amino acids, and amino acidanalogs in general. Furthermore, the amino acid can be D (dextrorotary)or L (levorotary).

In some embodiments, two DNA polymerase are joined via a linking group.The linking group can be a chemical crosslinking agent, including, forexample, succinimidyl-(N-maleimidomethyl)-cyclohexane-1-carboxylate(SMCC). The linking group can also be an additional amino acidsequence(s), including, for example, a polyalanine, polyglycine orsimilarly, linking group.

Alternatively, in some embodiments, the coding sequences of each DNApolymerase in the polypeptide are directly joined and expressed as afusion protein. Alternatively, an amino acid linker sequence may also beencoded in the polypeptide coding sequence and employed to separate thefirst and second polypeptide components by a distance sufficient toensure that each polypeptide folds into its secondary and tertiarystructures. Such an amino acid linker sequence is incorporated into thefusion protein using recombinant techniques well known in the art.Suitable peptide linker sequences may be chosen based on the followingfactors: (1) their ability to adopt a flexible extended conformation;(2) their inability to adopt a secondary structure that could interactwith functional epitopes on the first and second polypeptides; and (3)the lack of hydrophobic or charged residues that might react with thepolypeptide functional epitopes. Typical peptide linker sequencescontain Gly, Ser, Val and Thr residues. Other near neutral amino acids,such as Ala can also be used in the linker sequence. Amino acidsequences which may be usefully employed as linkers include thosedisclosed in Maratea et al. (1985) Gene 40:39-46; Murphy et al. (1986)Proc. Natl. Acad. Sci. USA 83:8258-8262; U.S. Pat. Nos. 4,935,233 and4,751,180. The linker sequence may generally be from 1 to about 50 aminoacids in length, e.g., 3, 4, 6, or 10 amino acids in length, but can be100 or 200 amino acids in length. Linker sequences may not be requiredwhen the second polypeptides have non-essential N-terminal amino acidregions that can be used to separate the functional domains and preventsteric interference.

IV. Expression and Purification

Nucleic acids encoding the DNA polymerases can be obtained using routinetechniques in the field of recombinant genetics. Basic texts disclosingthe general methods of use in this invention include Sambrook andRussell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001);Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); andCurrent Protocols in Molecular Biology (Ausubel et al., eds.,1994-1999). Such nucleic acids may also be obtained through in vitroamplification methods such as those described herein and in Berger,Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No.4,683,202; PCR Protocols A Guide to Methods and Applications (Innis etal., eds) Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim& Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991)3: 81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173;Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell etal. (1989) J. Clin. Chem., 35: 1826; Landegren et al., (1988) Science241: 1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu andWallace (1989) Gene 4: 560; and Barringer et al. (1990) Gene 89: 117,each of which is incorporated by reference in its entirety for allpurposes and in particular for all teachings related to amplificationmethods.

One of skill will recognize that modifications can additionally be madeto the DNA polymerases without diminishing their biological activity.Some modifications may be made to facilitate the cloning, expression, orincorporation of a domain into a fusion protein. Such modifications arewell known to those of skill in the art and include, for example, theaddition of codons at either terminus of the polynucleotide that encodesthe binding domain to provide, for example, a methionine added at theamino terminus to provide an initiation site, or additional amino acids(e.g., poly His) placed on either terminus to create convenientlylocated restriction sites or termination codons or purificationsequences.

The fusion polypeptides as described herein can be expressed in avariety of host cells, including E. coli, other bacterial hosts, yeasts,filamentous fungi, and various higher eukaryotic cells such as the COS,CHO and HeLa cells lines and myeloma cell lines. Techniques for geneexpression in microorganisms are described in, for example, Smith, GeneExpression in Recombinant Microorganisms (Bioprocess Technology, Vol.22), Marcel Dekker, 1994. Examples of bacteria that are useful forexpression include, but are not limited to, Escherichia, Enterobacter,Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus,Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus.Filamentous fungi that are useful as expression hosts include, forexample, the following genera: Aspergillus, Trichoderma, Neurospora,Penicillium, Cephalosporium, Achlya, Podospora, Mucor, Cochliobolus, andPyricularia. See, e.g., U.S. Pat. No. 5,679,543 and Stahl and Tudzynski,Eds., Molecular Biology in Filamentous Fungi, John Wiley & Sons, 1992.Synthesis of heterologous proteins in yeast is well known and describedin the literature. Methods in Yeast Genetics, Sherman, F., et al., ColdSpring Harbor Laboratory, (1982) is a well-recognized work describingthe various methods available to produce the enzymes in yeast.

There are many expression systems for producing the polypeptides thatare well known to those of ordinary skill in the art. (See, e.g., GeneExpression Systems, Fernandex and Hoeffler, Eds. Academic Press, 1999;Sambrook and Russell, supra; and Ausubel et al, supra.) Typically, thepolynucleotide that encodes the polypeptide is placed under the controlof a promoter that is functional in the desired host cell. Manydifferent promoters are available and known to one of skill in the art,and can be used in the expression vectors of the invention, depending onthe particular application. Ordinarily, the promoter selected dependsupon the cell in which the promoter is to be active. Other expressioncontrol sequences such as ribosome binding sites, transcriptiontermination sites and the like are also optionally included. Constructsthat include one or more of these control sequences are termed“expression cassettes.” Accordingly, the nucleic acids that encode thejoined polypeptides are incorporated for high level expression in adesired host cell.

Expression control sequences that are suitable for use in a particularhost cell are often obtained by cloning a gene that is expressed in thatcell. Commonly used prokaryotic control sequences, which are definedherein to include promoters for transcription initiation, optionallywith an operator, along with ribosome binding site sequences, includesuch commonly used promoters as the beta-lactamase (penicillinase) andlactose (lac) promoter systems (Change et al., Nature (1977) 198: 1056),the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res.(1980) 8: 4057), the tac promoter (DeBoer, et al., Proc. Natl. Acad.Sci. U.S.A. (1983) 80:21-25); and the lambda-derived PL promoter andN-gene ribosome binding site (Shimatake et al., Nature (1981) 292: 128).The particular promoter system is not critical; any available promoterthat functions in prokaryotes and provides the desired level of activitycan be used. Standard bacterial expression vectors include plasmids suchas pBR322-based plasmids, e.g., pBLUESCRIPT™, pSKF, pET23D, lambda-phagederived vectors, and fusion expression systems such as GST and LacZ.Epitope tags can also be added to recombinant proteins to provideconvenient methods of isolation, e.g., c-myc, HA-tag, 6-His tag, maltosebinding protein, VSV-G tag, anti-DYKDDDDK tag, or any such tag, a largenumber of which are well known to those of skill in the art.

For expression in prokaryotic cells other than E. coli, a promoter thatfunctions in the particular prokaryotic species is required. Suchpromoters can be obtained from genes that have been cloned from thespecies, or heterologous promoters can be used. For example, the hybridtrp-lac promoter functions in Bacillus sp. in addition to E. coli. Theseand other suitable bacterial promoters are well known in the art and aredescribed, e.g., in Sambrook et al. and Ausubel et al. Bacterialexpression systems for expressing the proteins of the invention areavailable in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.,Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kitsfor such expression systems are commercially available.

Eukaryotic expression systems for mammalian cells, yeast, and insectcells are well known in the art and are also commercially available. Inyeast, vectors include Yeast Integrating plasmids (e.g., YIp5) and YeastReplicating plasmids (the YRp series plasmids) and pGPD-2. Expressionvectors containing regulatory elements from eukaryotic viruses aretypically used in eukaryotic expression vectors, e.g., SV40 vectors,papilloma virus vectors, and vectors derived from Epstein-Barr virus.Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the CMV promoter, SV40 earlypromoter, SV40 later promoter, metallothionein promoter, murine mammarytumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter,or other promoters shown effective for expression in eukaryotic cells.

Either constitutive or regulated promoters can be used. Regulatedpromoters can be advantageous because the host cells can be grown tohigh densities before expression of the fusion polypeptides is induced.High level expression of heterologous proteins slows cell growth in somesituations. An inducible promoter is a promoter that directs expressionof a gene where the level of expression is alterable by environmental ordevelopmental factors such as, for example, temperature, pH, anaerobicor aerobic conditions, light, transcription factors and chemicals.

For E. coli and other bacterial host cells, inducible promoters areknown to those of skill in the art. These include, for example, the lacpromoter, the bacteriophage lambda P_(L) promoter, the hybrid trp-lacpromoter (Amann et al. (1983) Gene 25: 167; de Boer et al. (1983) Proc.Nat'l. Acad. Sci. USA 80: 21), and the bacteriophage T7 promoter(Studier et al. (1986) J. Mol. Biol.; Tabor et al. (1985) Proc. Nat'lAcad. Sci. USA 82: 1074-8). These promoters and their use are alsodiscussed in Sambrook et al., supra.

Translational coupling may be used to enhance expression. The strategyuses a short upstream open reading frame derived from a highly expressedgene native to the translational system, which is placed downstream ofthe promoter, and a ribosome binding site followed after a few aminoacid codons by a termination codon. Just prior to the termination codonis a second ribosome binding site, and following the termination codonis a start codon for the initiation of translation. The system dissolvessecondary structure in the RNA, allowing for the efficient initiation oftranslation. See Squires, et. al. (1988), J. Biol. Chem. 263:16297-16302.

The construction of polynucleotide constructs generally requires the useof vectors able to replicate in bacteria. Such vectors are commonly usedin the art. A plethora of kits are commercially available for thepurification of plasmids from bacteria (for example, EasyPrep™,FlexiPrep™, from Pharmacia Biotech; StrataClean™, from Stratagene; and,QIAexpress® Expression System, Qiagen). The isolated and purifiedplasmids can then be further manipulated to produce other plasmids, andused to transform cells.

The polypeptides described herein can be expressed intracellularly, orcan be secreted from the cell. Intracellular expression often results inhigh yields. If necessary, the amount of soluble, active fusionpolypeptide may be increased by performing refolding procedures (see,e.g., Sambrook et al., supra.; Marston et al., Bio/Technology (1984) 2:800; Schoner et al., Bio/Technology (1985) 3: 151). Polypeptides can beexpressed in a variety of host cells, including E. coli, other bacterialhosts, yeast, and various higher eukaryotic cells such as the COS, CHOand HeLa cells lines and myeloma cell lines. The host cells can bemammalian cells, insect cells, or microorganisms, such as, for example,yeast cells, bacterial cells, or fungal cells.

Once expressed, the polypeptides can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, gel electrophoresis and thelike (see, generally, R. Scopes, Protein Purification, Springer-Verlag,N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to ProteinPurification., Academic Press, Inc. N.Y. (1990)). Substantially purecompositions of at least about 90 to 95% homogeneity are preferred, and98 to 99% or more homogeneity are most preferred. Once purified,partially or to homogeneity as desired, the polypeptides may then beused (e.g., as immunogens for antibody production).

To facilitate purification of the polypeptides, the nucleic acids thatencode the polypeptides can also include a coding sequence for anepitope or “tag” for which an affinity binding reagent is available.Examples of suitable epitopes include the myc and V-5 reporter genes;expression vectors useful for recombinant production of fusionpolypeptides having these epitopes are commercially available (e.g.,Invitrogen (Carlsbad Calif.) vectors pcDNA3.1/Myc-His andpcDNA3.1/V5-His are suitable for expression in mammalian cells).Additional expression vectors suitable for attaching a tag to the fusionproteins of the invention, and corresponding detection systems are knownto those of skill in the art, and several are commercially available(e.g., FLAG″ (Kodak, Rochester N.Y.). Another example of a suitable tagis a polyhistidine sequence, which is capable of binding to metalchelate affinity ligands. Typically, six adjacent histidines are used,although one can use more or less than six. Suitable metal chelateaffinity ligands that can serve as the binding moiety for apolyhistidine tag include nitrilo-tri-acetic acid (NTA) (Hochuli, E.(1990) “Purification of recombinant proteins with metal chelatingadsorbents” In Genetic Engineering: Principles and Methods, J. K.Setlow, Ed., Plenum Press, N.Y.; commercially available from Qiagen(Santa Clarita, Calif.)).

One of skill in the art would recognize that after biological expressionor purification, the polymerase peptide (s) may possess a conformationsubstantially different than the native conformations of the constituentpolypeptides. In this case, it may be necessary or desirable to denatureand reduce the polypeptide and then to cause the polypeptide to re-foldinto the preferred conformation. Methods of reducing and denaturingproteins and inducing re-folding are well known to those of skill in theart (See, Debinski et al. (1993) J. Biol. Chem. 268: 14065-14070;Kreitman and Pastan (1993) Bioconjug. Chem. 4: 581-585; and Buchner etal. (1992) Anal. Biochem. 205: 263-270). Debinski et al., for example,describe the denaturation and reduction of inclusion body proteins inguanidine-DTE. The protein is then refolded in a redox buffer containingoxidized glutathione and L-arginine.

V. Methods of Use

Reverse transcription (RT) is an amplification method that copies RNAinto DNA. RT reactions can be performed with reaction mixtures asdescribed herein. For example, the invention provides for reversetranscribing one or more RNA (including for example, all RNA in a cell,e.g., to make a cDNA library) under conditions to allow for reversetranscription and generation of a first and optionally second strandcDNA. The RT reaction can be primed with a random primer, an oligo dT,or an RNA-specific primer. Components and conditions for RT reactionsare generally known.

If desired, the reactions can further comprise RT-PCR. Standardtechniques for performing PCR assays are known in the art (PCRTechnology: Principles and Applications for DNA Amplification (Erlich,ed., 1989); PCR Protocols: A Guide to Methods and Applications (Innis,Gelfland, Sninsky, &, White, eds., 1990); Mattila et al., Nucleic AcidsRes. 19: 4967 (1991); Eckert & Kunkel, PCR Methods and Applications 1:17 (1991); Wallace et al., Ligase Chain Reaction, in Technologies forDetection of DNA Damage and Mutations, pp. 307-322 (Pfiefer, ed.,1996)). RT and PCR reactions are often used in the same assay and arereferred to as RT-PCR. RT-PCR combines reverse transcription of RNA intoDNA and subsequent DNA amplification reactions in a single reaction.Optimal reverse transcription, hybridization, and amplificationconditions will vary depending upon the sequence composition andlength(s) of the primers and target(s) employed, and the experimentalmethod selected by the practitioner. Various guidelines may be used toselect appropriate primer sequences and hybridization conditions (see,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.)(1989); Ausubel, F. M. et al., eds. (1999) Short Protocols in MolecularBiology, 4th edition, John Wiley & Sons); Ausubel, F. M. et al., eds.(1999-2010) Current Protocols in Molecular Biology, John Wiley & Sons).

The practice of the present invention can employ conventional methods ofchemistry, biochemistry, molecular biology, cell biology, genetics,immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Gennaro, A.R., ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., MackPublishing Co.; Hardman, J. G., Limbird, L. E., and Gilman, A. G., eds.(2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-HillCo.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press,Inc.; Weir, D. M., and Blackwell, C. C., eds. (1986) Handbook ofExperimental Immunology, Vols. I-IV, Blackwell Scientific Publications;Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual,2nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al., eds. (1999-2010) Current Protocols in Molecular Biology,John Wiley & Sons; Ream et al., eds. (1998) Molecular BiologyTechniques: An Intensive Laboratory Course, Academic Press; Newton, C.R., and Graham, A., eds. (1997) PCR (Introduction to BiotechniquesSeries), 2nd ed., Springer Verlag; Sambrook et al., Molecular Cloning: ALaboratory Manual (2nd ed.) (1989).

An advantage of the reaction mixtures of the invention is that thereaction mixtures allow for use of a reverse transcriptase in an RT orDNA-dependent DNA polymerase reaction at a higher temperature than wouldotherwise be possible. Thus, in embodiments, the dimeric or trimericenzymes described herein can be used at, 37° or 42° C., or a temperaturegreater than 42° C., for example, between 43°-55°, 45°-56°, 45°-65° C.,etc. Higher temperature RT reactions are particularly helpful insituations where the template RNA forms secondary structure at normal RTtemperatures (e.g., 37° or 42° C.) that partially or completely inhibitreverse transcription.

In cases in which the polypeptide as described herein comprises one ortwo DNA-dependent DNA polymerases, the polypeptide can be used in aprimer extension reaction in a template-dependent manner. In someembodiments, the primer extension reaction is an amplification reaction.In some embodiments, the amplification reaction is monitored inreal-time and optionally is used to quantify the initial amount oftarget nucleic acid.

Such amplification reactions include without limitation polymerase chainreaction (PCR), DNA ligase chain reaction (LCR), and RNAtranscription-based (such as TAS and 3 SR) amplification reactions aswell as others known to those of skill in the art. Polymerase chainreactions that can be conducted using the compositions described hereininclude without limitation reverse-transcription PCR (rt-PCR) andquantitative PCR (qPCR).

In some embodiments, dye-based qPCR detection methods are used tomonitor amplification reactions utilizing components of the invention.Such detection methods generally rely on monitoring the increase influorescence signal due to the binding of DNA-binding dye to theamplified DNA. For example, SYBR Green I, a commonly used fluorescentDNA binding dye, binds all double-stranded DNA and detection ismonitored by measuring the increase in fluorescence throughout thecycle. SYBR Green I has an excitation and emission maxima of 494 nm and521 nm, respectively.

In other embodiments, probe-based qPCR detection methods are used tomonitor amplification reactions utilizing components of the invention.Such detection methods generally rely on the sequence-specific detectionof a desired PCR product. Unlike dye-based qPCR methods that detect alldouble-stranded DNA, probe-based qPCR utilizes a fluorescent-labeledtarget-specific probe, which detects specific sequences in the amplifiedDNA.

VI. Reaction Mixtures

Reaction mixtures comprising the polypeptides described herein areprovided. The reaction mixtures can comprise, for example, a targetnucleic acid, e.g., an RNA target where reverse transcription is to takeplace, or DNA where a DNA-dependent polymerase reaction is to takeplace. The reaction mixtures can comprise appropriate nucleotides (e.g.,deoxynucleotides (dNTPs) or dideoxynucleotides) and in some embodiments,at least one buffer. Exemplary buffers can include, for example andwithout limitation, Tris, HEPES, ACES, PIPES, MOPSO, BES, MOPS, TES,TAPSO, POPSO, BICINE, TAPS, or AMPSO. The reaction mixtures canoptionally comprise one or more oligonucleotides that function as aprimer for template-dependent nucleic acid extension, one or moreoligonucleotides that function as a probe (e.g., linked to a label suchas a quencher, fluorescent dye, etc.), and/or a double stranded DNAbinding dye (e.g., SYBRGREEN). In some embodiments, the reaction mixturewill further comprises a separate DNA-dependent DNA polymerase. In someembodiments, the reaction mixture will further comprises magnesium(Mg++).

VII. Kits

In one aspect, kits for conducting nucleic acid extension (andoptionally cyclic amplification, e.g., such as PCR) reactions areprovided. In some embodiments, such kits include polymerases, andoptionally dNTPs, and at least one buffer. Such kits may also includestabilizers and other additives (e.g., heparin and/or sarcosine) toincrease the efficiency of the amplification reactions. Such kits mayalso include one or more primers as well as instructions for conductingnucleic acid amplification reactions using the components of the kits.In some embodiments, the kits will further comprises a separateDNA-dependent DNA polymerase.

Examples Methods and Materials Cloning and Expression:

MLV reverse transcriptase gene was cloned into the pET28 vector by PCRto amplify the gene and followed by restriction enzyme digestion andligation. The expression construct contained a gene encoding a histidinetag that was fused to the N-terminus of the reverse transcriptase. TwoMLV genes were linked together through a linker sequence that containsrestriction sites HindIII, NheI, and SacI.

The expression plasmid was transformed into E. coli BL21/DE3 cells. Thecells containing the expression plasmid were inoculated for overnightculturing in LB medium, 1/100 of the culture was used to seed a fresh LBmedium the following morning. Cells were grown at 16 C. After celldensity reached OD600=0.6 to 0.8, IPTG was added to 0.1 mM to induce theprotein expression. Cells were collected 15 hrs after induction.

MLV RT aa sequence - SEQ ID NO: 1:TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL FLV RT aa sequence - SEQ ID NO: 2:TLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTIL MM Dimeric RT sequence(linker underlined) - SEQ ID NO: 3:EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSS SASMNM Dimeric RT sequence(linker underlined) - SEQ ID NO: 4:EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSS SASMNMN Dimeric RT sequence(linker underlined) - SEQ ID NO: 5:EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSS SASFF Dimeric RT sequence(linker underlined) - SEQ ID NO: 6:EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASFNF Dimeric RT sequence(linker underlined) - SEQ ID NO: 7:EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASFNFN Dimeric RT sequence(linker underlined) - SEQ ID NO: 8:EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASFM Dimeric RT sequence(linker underlined) - SEQ ID NO: 9:EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASFNM Dimeric RT sequence(linker underlined) - SEQ ID NO: 10:EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASFNMN Dimeric RT sequence(linker underlined) - SEQ ID NO: 11:EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRNILDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASMF Dimeric RT sequence(linker underlined) - SEQ ID NO: 12:EFTLNIEDEHRLHETSKEPDVSLG STWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASMNF Dimeric RT sequence(linker underlined) - SEQ ID NO: 13:EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASMNFN Dimeric RT sequence(linker underlined) - SEQ ID NO: 14:EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASFFN Dimeric RT sequence(linker underlined) - SEQ ID NO: 15:EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASFMN Dimeric RT sequence(linker underlined) - SEQ ID NO: 16:EFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTDGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASMFN Dimeric RT sequence (linker underlined) - SEQ ID NO: 17:EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGMAHCQAPVLIQLKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPVKKPGTKDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDLKDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDEALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKGYRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNPRQVREFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFENIRKALLSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDTVASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKWLSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGKPPRLSPDLAETMAQTDLTDQPLPDADLTWYTNGSSFIRNGERKAGAAVTTESEVIWAASLPPGTSAQRAELIALTQALKMAKGKKLTVYTDSRYAFATAHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRLADDTAKKAATETQSSLTILSSSASMMN Dimeric RT sequence (linker underlined) - SEQ ID NO: 18:EFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSSSASKLEFTLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTNGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLSS SAS

Enzyme Purification:

E. coli cells expressed the enzyme were re-suspended in a lysis buffer.Cells were disrupted on ice with pulses of sonication (2×30 sec, dutycycle 80%, output 10), and centrifuged at 19,000 rpm for 10 min. Thesupernatant was filtered through a 0.22 um filtration unit, diluted with10× buffer A for iMAC chromatography (50 mM KPO4, pH6.5, 300 mM KCl,0.1% Tween 20), and loaded onto a Nuvia iMAC column. The enzyme waseluted with a linear gradient to buffer B (buffer A plus 500 mMimidazole). The fraction containing the enzyme was pooled andconcentrated with an Amicon Ultra-15 centrifugal filter unit. The samplewas applied to a size exclusion column (SEC650), the fractionscontaining the purified enzyme was collected.

Rt-Qpcr Assay:

iScript Advanced reverse transcription mix was used for the reversetranscription of cDNA. The purified enzyme was added into the reaction.iScript advanced reverse transcriptase was used as a control. In orderto study the effect of temperature on cDNA synthesis the reversetranscription reaction was carried out at 42, 55, and 60 C for 30 minrespectively. The cDNA synthesis speed by the purified enzymes was alsocompared to the iScript Advanced reverse transcriptase, in which thereverse transcription was carried out in iScript Advanced reaction mixfor 30, 2, 1 min respectively. After the cDNA synthesis, qPCR wasperformed on different targets. To assess the performance of the cDNAsynthesis, delta quantification cycle (dCq) was calculated using a cDNAreaction at 42 C for 30 min as a control.

TABLE 1 Dimeric MLV RTs (MNM and MM) displayed better thermostabilitythan the wild type control. Two-step RT-qPCR was performed on 6different targets. MNM MM Control MNM MM Control MNM MM Control APC CBPbeta Actin Cq 42 C. 25.19 26.99 25.17 26.65 27.20 26.78 16.20 16.8916.46 55 C. 26.26 26.75 26.30 27.74 28.27 29.21 17.32 17.71 19.23 60 C.26.21 27.21 26.73 27.65 28.38 29.52 17.26 17.56 19.38 dCq 55 C.-42 C.1.07 −0.24 1.13 1.10 1.07 2.43 1.12 0.82 2.77 60 C.-42 C. 1.01 0.22 1.561.00 1.19 2.74 1.06 0.67 2.92 GAPDH 18 s Tub Cq 42 C. 16.59 17.45 16.164.58 5.15 4.88 18.61 19.23 18.49 55 C. 17.13 18.48 18.62 10.54 10.8914.08 19.37 20.09 21.14 60 C. 17.15 18.68 19.24 10.96 11.45 14.68 19.2419.92 21.53 dCq 55 C.-42 C. 0.54 1.02 2.46 5.96 5.74 9.20 0.76 0.86 2.6460 C.-42 C. 0.56 1.22 3.08 6.38 6.30 9.80 0.63 0.69 3.03

TABLE 2 Additional dimeric RTs displayed better thermostability than thewild type control. The template target was human GAPDH. RT MF MNF MM MNMFNM control Cq (55 C.) 20.64 20.88 16.91 16.64 21.63 17.16 Cq (42 C.)19.60 19.70 15.99 15.86 20.75 13.28 dCq 1.04 1.18 0.92 0.78 0.88 3.88 Cq(60 C.) 21.32 20.79 17.58 17.35 21.54 19.40 Cq (42 C.) 18.41 18.39 14.6714.65 19.66 13.30 dCq 2.91 2.39 2.91 2.69 1.88 6.10 Cq (42 C. 5 min)18.63 18.62 14.63 14.55 19.53 16.06 Cq (42 C. 30 min) 18.41 18.39 14.6714.65 19.66 13.30 dCq 0.22 0.23 −0.04 −0.11 −0.13 2.76 Cq (42 C. 2.5min) 19.54 19.60 15.35 15.19 20.41 17.00 Cq (42 C. 30 min) 18.41 18.3914.67 14.65 19.66 13.30 dCq 1.13 1.21 0.68 0.53 0.75 3.70 MF:MLVRT-FLVRT dimer; MNF: MLVRT RNaseH(−)-FLVRT dimer; MM: MLVRT-MLVRTdimer; MNM: MLVRT RNaseH(−)-MLVRT dimer; FNM: FLVRT RNaseH(−)-MLVRTdimer; RT control: Purified monomeric RT.

All five dimeric RT's exhibited better stability than the monomeric RTcontrol at the two temps, as shown for example by the lower dCq values.All five dimeric RTs also exhibited reduced reaction time vs the RTcontrol.

The examples and embodiments described herein are for illustrativepurposes only and that various modifications or changes in light thereofwill be suggested to persons skilled in the art and are to be includedwithin the spirit and purview of this application and scope of theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes.

1. A nucleic acid encoding a polypeptide comprising a first reversetranscriptase and a second reverse transcriptase covalently linked by aheterologous linker, wherein the first reverse transcriptase and thesecond reverse transcriptase are at least 90% identical to one another.2. The nucleic acid according to claim 1, wherein the first reversetranscriptase and second reverse transcriptase are identical.
 3. Thenucleic acid according to claim 1, wherein said heterologous linker isbetween 1-30 amino acids long.
 4. The nucleic acid according to claim 1,wherein the first or second reverse transcriptase is selected from thegroup consisting of murine leukemia virus (MLV) reverse transcriptase,Feline leukemia virus (FLV) reverse transcriptase, bovine leukemia virus(BLV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reversetranscriptase, Respiratory Syncytial Virus (RSV) reverse transcriptase,Equine Infectious Anemia Virus (EIAV) reverse transcriptase,Rous-associated Virus-2 (RAV2) reverse transcriptase, SUPERSCRIPT IIreverse transcriptase, SUPERSCRIPT I reverse transcriptase, THERMOSCRIPTreverse transcriptase and MMLV RNase H— reverse transcriptase.
 5. Thenucleic acid according to claim 1, wherein the encoded polypeptide is nomore than 2000 amino acids in length.
 6. The nucleic acid according toclaim 1, wherein the first or second reverse transcriptase has at leastone mutation compared to a naturally-occurring reverse transcriptase. 7.The nucleic acid according to claim 6, wherein the first or secondreverse transcriptase is an RNase H— reverse transcriptase or comprisesSEQ ID NO:
 1. 8. The nucleic acid according to claim 1, said nucleicacid encoding a polypeptide comprising a first reverse transcriptase anda second reverse transcriptase covalently linked by a heterologouslinker, wherein the first reverse transcriptase and the second reversetranscriptase are at least 90% identical.
 9. The nucleic acid accordingto claim 8, wherein the first and second reverse transcriptase are amurine leukemia virus (MLV) reverse transcriptase.
 10. The nucleic acidaccording to claim 8, wherein the first and second reverse transcriptaseare a Feline leukemia virus (FLV) reverse transcriptase.
 11. The nucleicacid according to claim 8, wherein the heterologous linker is between1-30 amino acids long.
 12. The nucleic acid according to claim 8,wherein the first or second reverse transcriptase is selected from thegroup consisting of murine leukemia virus (MLV) reverse transcriptase,Feline leukemia virus (FLV) reverse transcriptase, bovine leukemia virus(BLV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reversetranscriptase, Respiratory Syncytial Virus (RSV) reverse transcriptase,Equine Infectious Anemia Virus (EIAV) reverse transcriptase,Rous-associated Virus-2 (RAV2) reverse transcriptase, SUPERSCRIPT IIreverse transcriptase, SUPERSCRIPT I reverse transcriptase, THERMOSCRIPTreverse transcriptase and MMLV RNase H— reverse transcriptase.
 13. Thenucleic acid according to claim 8, wherein the encoded polypeptide is nomore than 2000 amino acids in length.
 14. The nucleic acid according toclaim 8, wherein the first or second reverse transcriptase has at leastone mutation compared to a naturally-occurring reverse transcriptase.15. The nucleic acid according to claim 14, wherein the first or secondreverse transcriptase is an RNase H— reverse transcriptase or comprisesSEQ ID NO:
 1. 16. The nucleic acid according to claim 8, wherein theencoded polypeptide comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, orSEQ ID NO:18.
 17. A polynucleotide vector comprising the nucleic acidaccording to claim
 1. 18. A host cell comprising the polynucleotidevector of claim
 17. 19. A method of producing a polypeptide comprisingculturing a host cell according to claim 18 and expressing saidpolypeptide, the polypeptide comprising a first reverse transcriptaseand a second reverse transcriptase covalently linked by a heterologouslinker, wherein the first reverse transcriptase and the second reversetranscriptase are identical to one another or are at least 90% identicalto one another.
 20. A method of performing a polymerase reactioncomprising contacting a target nucleic acid with a polypeptidecomprising a first reverse transcriptase and a second reversetranscriptase covalently linked by a heterologous linker, wherein thefirst reverse transcriptase and the second reverse transcriptase areidentical to one another or are at least 90% identical to one anotherand an oligonucleotide primer that hybridizes to the target nucleic acidunder conditions to allow the polypeptide to extend the oligonucleotideprimer in an template-dependent manner, thereby performing a polymerasereaction.